4. DNA Extraction, Detection and Quantification

What is the first step in molecular testing involving nucleic acids?

To extract DNA or RNA from the sample and purify it.

What are the four basic steps in DNA extraction?

1) Collection of the sample, 2) Lysis of the cell, 3) Removal of membrane fragments, proteins, and RNA, 4) Concentration of purified DNA.

What is the purpose of EDTA in blood sample collection?

EDTA prevents clotting and inhibits enzyme activity by chelating ions like Mg2+.

Which anticoagulant should NOT be used for DNA extraction and why?

Heparin, because it can interfere with molecular methods and inhibit Taq polymerase in PCR.

What types of specimens can DNA be extracted from?

Whole blood, bone marrow, buffy coat, fresh/frozen tissue, tumors, skin, fetal chorionic villi, umbilical cord, FFPE tissue, saliva, amniotic fluid, cultured cells.

How should whole blood and bone marrow be stored after collection?

At room temperature for up to 4 days.

What is the recommended storage temperature for DNA long term?

-80 °C.

What is the recommended storage methods for FFPE tissue?

It can be stored indefinitely.

What is the significance of the 260/280 absorbance ratio in nucleic acid quantification?

A 260/280 ratio of 1.8 is considered pure for DNA, and 2.0 for RNA.

What does a 260/230 ratio < 1.8 indicate?

DNA is contaminated with organic material or carbohydrates.

What happens to nucleic acids when precipitation with ethanol occurs?

They precipitate out of solution due to the less polar solvent properties of ethanol.

What are common methods for DNA extraction mentioned in the notes?

Phenol/chloroform method, column methodology, and magnetic bead extraction.

What should be used to avoid introducing nucleases into the sample?

Filtered pipette tips.

What is the risk of introducing nucleases during DNA extraction?

Nucleases can degrade DNA and RNA.

What is the purpose of using proteinase K in sample preparation?

To digest the lysate and help extract DNA.

How should buffy coat samples be stored for later use?

They should be stored frozen.

What is the function of NH4Cl in whole blood preparation?

To lyse red blood cells while preserving the nuclear membrane.

What is a common method for lysing cells during DNA extraction?

Using a lysis buffer.

What is one disadvantage of using spectrophotometry for nucleic acid quantification?

It cannot distinguish between DNA and RNA.

How is DNA typically isolated from lysates using magnetic beads?

By binding to silica surfaces of the beads in the presence of chaotropic salt.

What temperature is recommended for RNA storage?

-80 °C.

How can RNA degradation be prevented during extraction?

By using RNase-free reagents and equipment.

Why is fluorescence-based nucleic acid measurement considered sensitive?

Because it can accurately measure low concentrations (pg/mL) even in the presence of contaminants.

What is the primary factor determining the extraction method used?

The type of specimen being processed.

What should be done if the 260/280 ratio is < 1.7?

Re-extract the DNA due to protein contamination.

What does RNAse do?

RNases degrade RNA into smaller components.

What is the importance of the house-keeping gene in quantitative PCR?

It helps infer the amount of amplifiable DNA present in a sample.

What safety measures should be taken when using phenol/chloroform?

Avoid contact with skin and eyes, and use in a fume hood.

What does a high molecular weight band on a gel indicate?

Intact DNA.

What method is often used to clean laboratory environments for RNA work?

Cleaning with RNase-destroying solutions.

What happens to DNA during storage if proper conditions are not maintained?

DNA can degrade over time.

How is DNA eluted off of a silica membrane in column extraction?

By changing buffering conditions.

What is the key principle behind the salt extraction method?

Cells are lysed with anionic detergent in the presence of a DNA stabilizer.

What does the law of exploits or contamination amid spectrophotometry relate to?

Any contaminants will alter the absorbance readings.

What is the consequence of having fragmented DNA?

It will absorb light at 260nm the same as unfragmented DNA.

How should amniotic fluid be processed for DNA extraction?

It should be centrifuged, and the cell pellet must be resuspended in lysis solution.

What is commonly added to the lysis buffer to help prevent RNase activity?

β-mercaptoethanol or DTT.

What is the impact of vortexing on DNA samples?

It can shear DNA, leading to degradation.

Why is proper specimen handling crucial for DNA extraction?

Improper handling can lead to DNA degradation.

What happens during the centrifugation of whole blood to prepare buffy coat?

The blood separates into layers, allowing for the isolation of the buffy coat.

What is the purpose of using a biosafety cabinet during sample preparation?

To maintain a controlled environment and reduce contamination risks.

What is the significance of generating a calibration curve in fluorescence quantitation?

It allows for accurate measurement of nucleic acids against known standards.

What type of substances can interfere with downstream tests when added as sample stabilizers?

Certain chemical stabilizers could affect the results of subsequent molecular tests.

What factor influences the storage condition for cultured cells?

Cultured cells are viable for up to 8 weeks with proper care.

What is the protocol for collecting saliva for DNA extraction?

Collect saliva in a tube, centrifuge, and resuspend the pellet in PBS.